Imp Dehydrogenase 2 Drives Aberrant Nucleolar Activity And-Books Pdf

IMP dehydrogenase 2 drives aberrant nucleolar activity and
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Articles Nature Cell Biology, a b GBM orthotopic Cut with vibratome Culture in. implantation 200 m slice dish, GBM tissue from GBM PDX mouse infused with C6 glucose for 6 h. Metabolic turnover Intracellular Biosynthetic, rate nucleotide levels rate. 0 08 100 MS imaging, Intracellular nucleotide, Metabolic turnover. H E GTP M 6 ATP M 6, nmol g 1 6 h 1, Biosynthetic rate.
level nmol g 1, 0 04 50 2 GBM, ATP GTP ATP GTP ATP GTP. c Primary glia U87MG LN229, Metabolic turnover rate Intracellular nucleotide levels Biosynthetic rate. P 0 0001 P 0 0001 P 0 0001, 0 6 60 000 25 000 P 0 0008. Intracellular nucleotide level, Relative turnover rate. amol cell 1 4 h 1, Biosynthetic rate, amol cell 1, P 0 0001 P 0 0001 P 0 0001 P 0 0001.
0 3 30 000 12 500, P 0 0001 P 0 0001 P 0 0006, P 0 0001 P 0 0001 P 0 0057. P 0 0003 P 0 045, ATP GTP UTP CTP ATP GTP UTP CTP ATP GTP UTP CTP. Fig 1 Upregulation of GTP biosynthesis in human and murine GBM a The metabolic turnover of GTP is higher than that of the other nucleotides in the. GBM PDX mouse model The metabolic turnover rate of the indicated nucleotides is calculated on the basis of the intracellular pool size middle and. biosynthetic rate right of ribonucleotides in the GBM tissues infused with U 13C glucose for 6 h H E haematoxylin and eosin b A murine GBM brain. section was treated with U 13C glucose and subjected to MS imaging analysis M 6 GTP was detected in organotypic GBM but not in normal brain tissue. while M 6 ATP was detected in both GBM and normal brain tissue Representative images are shown from two independent experiments Scale bar. 300 m c The metabolic turnover of GTP is higher than that of the other nucleotides in GBM cells but not in primary glia The metabolic turnover rate of. the indicated nucleotides is calculated on the basis of the intracellular pool size middle and biosynthetic rate bottom of ribonucleotides in the. indicated cells labelled with U 13C glucose for 4 h The data are presented as mean s d n 3 biologically independent samples One way analysis of. variance ANOVA, Consistent with the results from GBM tissues in cultured GBM IMP for GTP biosynthesis Fig 2b displayed the largest increase. cells the pool size and the turnover rate of GTP were elevated com relative to control brains about twofold P 0 05 In contrast the. pared to those of ATP UTP and CTP Fig 1c and Supplementary expression levels of the isoenzyme encoded by Impdh1 which has. Fig 1b c In contrast the turnover rate of GTP in less prolifera indistinguishable substrate affinities and catalytic activities21 23 was. tive primary glia is comparable to those of ATP and CTP and less minimally affected or even decreased. than that of UTP Both GBM and primary glia allocate a substantial Although both the association of increased IMPDH activity with. amount of ATP for biosynthesis of nicotinamide adenine dinucleo tumours24 and the anti tumour activity of IMPDH inhibition have. tide NAD and S adenosylmethionine SAM Supplementary been noted for over 50 years ago25 30 the primary role of IMPDH in. Fig 1d Similar results were observed in neural stem cells NSCs promoting tumour growth has remained unclear To investigate the. and the isogenic GBM stem cells GSCs that are responsible for potential primary role for IMPDH we first surveyed the expression. therapeutic resistance15 18 Supplementary Fig 1e Detailed quanti of IMPDH2 in human GBM patients Five human GBM databases. fication showed that the biosynthetic rate of SAM and NAD from revealed a more than twofold upregulation of IMPDH2 mRNA. ATP pathways was more than twofold higher in U87MG and LN229 Supplementary Fig 2b Another key enzyme for de novo GTP. than in primary glia consistent with increased growth demand in biosynthesis GMPS was also upregulated and showed a high cor. GBM19 20 We also found that primary glia and NSCs compared relation with IMPDH2 expression levels Supplementary Fig 2b c. with GBM and GSCs possess not only more active uptake of Interestingly expression of HPRT1 mRNA an essential gene for the. purine metabolites from media but also greater active salvage GTP salvage GTP pathway was downregulated while APRT and ADK. biosynthesis Supplementary Fig 1f j These results suggest that mRNA critical genes for the salvage ATP pathway were reciprocally. glucose metabolism in GBM is reprogrammed to increase de novo upregulated showing significant correlation with IMPDH2 levels. GTP biosynthesis Supplementary Fig 2b c Immunodetection confirmed overex. pressed IMPDH2 in murine glioma human PDX GBM tissues. Upregulation of IMPDH2 in human and murine GBM established GBM cell lines and GSCs Fig 2c and Supplementary. To investigate the molecular mechanism underlying the increased Fig 3 Four glioma patient cohorts total 426 specimens showed a. GTP biosynthesis in GBM we analysed gene expression in ten positive correlation of IMPDH2 with increased glioma malignancy. genetically different mouse gliomas Fig 2a and Supplementary Fig 2d and negative correlation with patient survival Fig 2e. Fig 2a Expression of Impdh2 encoding the enzyme that commits While the mutational status of isocitrate dehydrogenase IDH is. 1004 Nature Cell Biology VOL 21 AUGUST 2019 1003 1014 www nature com naturecellbiology. Nature Cell Biology Articles, a Mouse strains, Normal GBM PDX tissue. Gfap driven brain Glioma, Nestin driven Anti IMPDH1 Anti IMPDH2.
Nestin driven, Ink4a Arf Pten fl fl 2, Combination Mutation in TERT promoter. PDGF A expression GBM4 TP53 and PIK3R1, PDGF B expression 0. p53 knockdown, amplification of MYC, NF1 knockdown. RNA profile, 5 2 5 0 2 5 Mutation in TERT promoter and. PC1 GBM8 PIK3R1 amplification of MYCN, log2 IMPDH2 mRNA levels.
11 0 PDGFRA and MDM2, Mutation in TERT promoter, Anti IMPDH2 TP53 and RB1. Normal GBM, ATP GTP Mutation in TERT promoter, Formate PRPP MPA GBM23 TSC1 APC PDGFRA and. PTEN CDKN2A loss, IMPDH2 GMPS, AMP SAMP IMP XMP GMP. Mutation in TERT promoter, APRT HPRT1 HPRT1 GBM29 TP53 RB1 and MAP3K1. Adenine Hypoxanthine Guanine FGFR3 TACC3 fusion, Adenosine Guanosine 50 m.
d Cohort no 2 e, Cohort no 1, P 0 0002 Cohort no 2 Cohort no 4. P 0 0009 High IMPDH2 IDH1 mutation, P 0 0035 P 0 0031. 100 Low IMPDH2 IDH1 wild type 100, progression free. Probability of, IMPDH2 low, IMPDH2 level, IMPDH2 level. IMPDH2 low, 50 IMPDH2 high, 1 1 IMPDH2 low, IMPDH2 high IMPDH2 high.
2 3 4 1 2 3 4 0 50 100 150 0 50 100 150, Grade Grade Months Months. Cohort no 3 Cohort no 4, Cohort no 3 IDH mutation, 3 3 100 IMPDH2 low. IMPDH2 level, IMPDH2 level, 50 IMPDH2 high, 2 3 4 2 3 4 0 500 1 000 1 500. Grade Grade Days, H E GBM tissue Anti IMPDH1 Anti IMPDH2. Fig 2 Upregulation of IMPDH2 in human and murine GBM a Principal component analysis in ten clinically relevant glioma mouse models by. Illumina cDNA microarray upper The black dots are control normal brain samples the red dots are glioma samples The experiment contains the. gene expression profiles from 13 normal and 59 glioma mice n 72 Impdh2 expression at mRNA levels lower left and immunohistochemical IHC. analysis of IMPDH2 in the Nestin driven Pdgf B in lnk4a arf Ptenfl fl mouse brain lower right The boxplots follow a Tukey style in which the lower. and upper hinges correspond to the first and third quartiles The IHC image is a representative one from four independent animals Scale bar 1 000 m. b A schematic diagram of the purine biosynthesis pathway c GBM tissue from the GBM PDX mouse expresses IMPDH2 while IMPDH1 protein was. undetectable n 1 experiment d Analysis of four cohorts showed increased IMPDH2 expression in human glioma specimens with different WHO. World Health Organization grades bottom H E staining of GBM and representative IHC for IMPDH1 and IMPDH2 The data are presented as. mean s e m n 91 for cohort 1 n 191 for cohort 2 n 91 for cohort 3 n 53 for cohort 4 One way ANOVA The IHC image is a representative one. from cohort 4 n 53 Scale bar 300 m e Kaplan Meier survival curves for three cohorts of glioma patients based on IDH mutational status cohort 1. n 90 cohort 3 n 38 and the relative strength of cytoplasmic IMPDH2 expression Cohort 4 n 32 is progression free survival and the others are. overall survival Log rank tests two sided were used for the statistical analysis. Nature Cell Biology VOL 21 AUGUST 2019 1003 1014 www nature com naturecellbiology 1005. Articles Nature Cell Biology, a Biosynthetic rate b Intracellular nucleotide levels c LN229.
GTP M 3 to M 10 GTP M 6 GTP GTP ATP, 7 600 P 0 0004 150. P 9 38 10 P 0 0078, Biosynthetic rate, Relative levels. Biosynthetic rate, Concentration, Concentration, 3 000 0 3 2 5. DMSO MPA 0 1 2 3 4 10 M MPA 0 4 0 4 h MPA 0 4 h MPA 0 4 h. Post labelling h Primary glia U87MG, ATP M 3 to M 10 ATP M 6 5 ATP. P 4 4 10 150 GTP ATP, Biosynthetic rate, Relative levels.
P 6 24 10 P 2 3 10, Biosynthetic rate, 14 000 100 1 0 6. Concentration, Concentration, 7 000 DMSO 50 0 5 3, DMSO MPA 0 1 2 3 4 10 M MPA 0 4 0 4 h MPA 0 4 h MPA 0 4 h. Post labelling h Primary glia U87MG, d e Biosynthetic rate. GTP GTP M 3 to M 10, P 0 0001 P 0 0001, P 0 0001 P 0 0001. P 0 0001 GTP M 6, CRISPR 1 0 12 000, Concentration mM.
Biosynthetic rate, Biosynthetic rate, 50 0 5 6 000 IMPDH1 KO. P 0 0006 500, 50 IMPDH2 KO, actin 0 IMPDH DKO, 40 1 2 3 4 h. Post labelling, ATP M 3 to M 10, 25 000 P 0 0001, 3 000 IMPDH2 KO. Biosynthetic rate, Biosynthetic rate, 1 500 IMPDH DKO. 0 1 2 3 4 h, Post labelling, Fig 3 IMPDH2 reprograms GTP metabolism in GBM a The biosynthetic rate of GTP but not that of ATP is decreased by pharmacological inhibition.
of IMPDH in U87MG cells The isotopomer distribution M 6 of GTP and ATP from U 13C glucose is shown on the right The data are presented as. mean s d n 3 biologically independent samples Unpaired two sided Student s t test b GTP levels were decreased by 4 h of MPA treatment in U87MG. cells n 1 experiment c Pharmacological inhibition of IMPDH activity leads to an acute decrease in the concentration of GTP but not ATP in GBM cells. The indicated cells were treated with 10 M MPA for 4 h and GTP and ATP concentrations were quantified by high performance liquid chromatography. HPLC for U251 cells and capillary electrophoresis CE MS for LN229 cells The data are presented as mean s d n 3 biologically independent samples. Unpaired two sided Student s t test d The indicated IMPDH KO U87MG cells were generated by the CRISPR Cas9 system as in the Methods The western. blot shows the upregulation of IMPDH1 in IMPDH2 KO U87MG and LN229 cells Non specific band is indicated by asterisk Cells were maintained with. 100 M guanosine supplemented medium which was replaced with DMEM 10 dialysed fetal bovine serum FBS medium 24 h before the assay GTP. levels were quantified by HPLC The data are presented as mean s d n 3 biologically independent samples One way ANOVA Western blot analysis. was performed at least twice e IMPDH2 KO and IMPDH DKO in U87MG cells decreased the biosynthetic rate of GTP The assay was performed as in. a The data are presented as mean s d n 3 biologically independent samples One way ANOVA. one of the most critical biomarkers for molecular classification IMPDH2 reprograms GTP metabolism in GBM. and patients are in general expected to survive longer16 18 31 high Next we assessed the impact of pharmacological and genetic inhi. IMPDH2 expression appeared to confer poor survival even in IDH bition of IMPDH2 Treatment with mycophenolic acid MPA. mutated glioma patients IMPDH1 levels were marginal in the a pan IMPDH inhibitor23 32 dramatically decreased de novo GTP. tested glioma specimens Fig 2c d and Supplementary Fig 3d g biosynthesis in GBM cells but not in primary glia Fig 3a c GTP. These results indicate that gliomagenesis is associated with upregu biosynthesis was moderately decreased in IMPDH1 knockout KO. lation of IMPDH2 but not IMPDH1 In summary the upregula U87MG cells whereas IMPDH2 KO markedly decreased GTP bio. tion of IMPDH2 and its association with systemic changes of genes synthesis Consequently double KO of both isotypes DKO led to a. critical for de novo and salvage GTP synthesis in GBM is consistent greater decrease in GTP biosynthesis and concentrations Fig 3d e. with a direct and primary effect of metabolic reprogramming of The results suggest that upregulation of IMPDH2 reprograms the. GTP biosynthesis on GBM nucleotide metabolism towards de novo GTP biosynthesis in GBM. 1006 Nature Cell Biology VOL 21 AUGUST 2019 1003 1014 www nature com naturecellbiology. Nature Cell Biology Articles, IMPDH2 upregulation promotes GBM proliferation in vitro that grow even faster than parental U87MG cells Supplementary. Although IMPDH2 KO decreased proliferation the cells were still Fig 5d Subsequently we observed significant upregulation of. responsive to MPA presumably owing to the compensation by IMPDH1 in IMPDH2 KO U87MG suggesting a mechanism. IMPDH1 Fig 4a In support of this notion IMPDH DKO further explaining the tumour growth Supplementary Fig 5d In contrast. decreased cell proliferation and sensitivity to MPA treatment Fig 4a IMPDH DKO significantly impaired tumour growth in the brain. and Supplementary Fig 4a To test this directly we compared dox Fig 4h corroborating the compensatory expression of IMPDH1. ycycline dependent ectopic expression of IMPDH1 or IMPDH2 in following IMPDH2 depletion Taken together these results reveal. Yuki Sugiura7 Oltea Sampetrean 8 Yoshiki Ikeda1 Mikako Warren9 10 Naoya Sakamoto2 Shuji Kitahara 11 Hirofumi Yoshino 1 Daisuke Yamashita 12 Kazutaka Sumita 1 Kara Wolfe 1 Lisa Lange 4 5 Satsuki Ikeda 3 Hiroko Shimada 1 Noriaki Minami 8 Akshiv Malhotra 1 Shin Morioka 2

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